MITOCHONDRIAL DYSFUNCTION, PERMEABILITY TRANSITION PORE, AND ACUTE PANCREATITIS Key words: acute pancreatitis, pancreatic acinar cell, mitochondria, permeability transition pore, lysosomes, cathepsin, autophagy, trypsin, vacuoles. Aims: Acute pancreatitis is a potentially fatal disease of exocrine pancreas, the pathogenesis of which remains obscure and specific treatments for which do not exist. Intra-acinar trypsinogen activation and formation of large vacuoles are key early pathologic responses of pancreatitis. Our recent studies revealed that acute pancreatitis causes profound impairment of autophagy, the main cellular degradative, lysosome-driven process. We showed that autophagy impairment results from defective, inefficient lysosomal protein degradation and mediates the accumulation of vacuoles and trypsin in acinar cells. On the other hand, we found that mitochondrial dysfunction, manifest by loss of the mitochondrial membrane potential (DYm), is a common response in models of acute pancreatitis. The principal mechanism of mitochondrial depolarization is through opening of permeability transition pore (PTP), a non-selective channel in the mitochondrial membrane. The overall goal of this proposal is to determine whether the dysfunctions of 2 critical organelles, the lysosomes and mitochondria, are linked;that is, whether PTP-dependent mitochondrial depolarization mediates lysosomal dysfunction, autophagy impairment, acinar cell vacuolization and trypsinogen activation in pancreatitis. We will use experimental in vivo and in vitro models of pancreatitis to determine PTP-mediated changes in DYm and mitochondrial reactive oxygen species (ROS), and the roles of PTP and mitochondrial ROS in the above-referred pathologic responses. For this purpose, we will apply genetic (knockout mice), molecular (transfections), and pharmacologic approaches. Our hypothesis states that PTP opening, which results in DYm loss and drop in mitochondrial ROS, mediates key pathologic responses of acute pancreatitis. Further, mitochondrial dysfunction mediates the defective lysosomal protein degradation and autophagy in pancreatitis, underlying acinar cell vacuolization and trypsinogen activation. An important mechanism linking the mitochondrial and lysosomal dysfunctions in pancreatitis is the PTP-mediated decrease in mitochondrial ROS. Based on our published and preliminary data for this application, we propose that PTP inactivation represents a promising novel therapeutic strategy to treat or mitigate the severity of acute pancreatitis. Research Plan: Specific Aims of our proposal are: (1). Determine the effect of PTP on DYm and ROS in in vivo and in vitro models of acute pancreatitis. (2). Determine the role of PTP in lysosomal dysfunction and impaired autophagy, underlying acinar cell vacuolization and trypsinogen activation in in vivo and in vitro models of pancreatitis.(3). Determine the role of mitochondrial ROS in lysosomal dysfunction, impaired autophagy, acinar cell vacuolization and trypsinogen activation in the in vitro model of pancreatitis. (4). Determine the effects of pharmacologic PTP inhibition on pathologic responses of acute pancreatitis. PUBLIC HEALTH RELEVANCE: Acute pancreatitis is a potentially fatal disease of exocrine pancreas, the pathogenesis of which remains unknown and specific treatments for which have not been developed. This disease is common in VA patient population. Our recent studies revealed that acute pancreatitis causes profound dysfunction of 2 critical cellular organelles, the mitochondria and the lysosomes. This proposal will use experimental models in mice and isolated cells to determine the molecular mechanisms of these dysfunctions, the link between them, and their roles in key pathologic responses of pancreatitis (such as accumulation of large vacuoles in pancreas and the premature, intrapancreatic activation of digestive enzymes). Further, based on the mechanism, we propose a novel therapeutic strategy to correct the mitochondrial and lysosomal dysfunctions and thus to treat or mitigate the severity of pancreatitis.